Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.11 (AMPK)
 12,425 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Metalloproteinases, such as collagenase or gelatinase, and their associated inhibitors appear to control connective tissue remodeling during follicular rupture. We examined the regulation of metalloproteinase inhibitor activity by various treatments in cultured rat granulosa cells. Granulosa cells were harvested from immature PMSG-primed rats and cultured with LH, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), cAMP, or forskolin. Inhibitor activity was measured in the medium. Increasing concentrations of either LH (0.1-1000 ng/ml) or TPA (2.5-100 nM) resulted in a dose-dependent increase in metalloproteinase inhibitor activity (2.9- and 2.4-fold increases above control, respectively). There was also a time-dependent induction of inhibitor activity in cells incubated in the presence of LH (100 ng/ml) for 6, 12, 18, or 24 h. Forskolin (0.1 mM) or cAMP (1 mM) treatment increased inhibitor activity 2.8- and 1.6-fold above that in control cultures. LH and TPA treatment in combination resulted in an additive increase in inhibitor activity compared to LH or TPA treatment alone. This finding suggested that the granulosa cell inhibitor activity might be induced through separate intracellular pathways. The inhibitor present in conditioned medium was isolated by chromatographic separation on a Sepharose 6B mol wt exclusion column. The inhibitor present was approximately 28,000 mol wt, which is consistent with the size of tissue inhibitor of metalloproteinase (TIMP). In addition to the granulosa cell experiments, changes in ovarian mRNA levels for TIMP were determined. There was a preovulatory increase in TIMP mRNA from whole rat ovaries, with the highest levels detected 12 h after hCG administration. The present study establishes that metalloproteinase inhibitor activity from rat granulosa cells is induced through separate pathways: a LH-cAMP-dependent protein kinase-A pathway and a cAMP-independent protein kinase-C pathway. Furthermore, a TIMP-like protein is observed in granulosa cell-conditioned medium, while TIMP mRNA is present in rat ovaries and increases before ovulation, suggesting that the granulosa cell metalloproteinase inhibitor is TIMP. We propose that TIMP acts in part to control the site and extent of follicular connective tissue remodeling associated with ovulation.
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PMID:Hormonal regulation of matrix metalloproteinase inhibitors in rat granulosa cells and ovaries. 184 3

Type beta transforming growth factors represent a family of polypeptides that modulate growth and differentiation. TGF-beta exerts its effects on target cells through interaction with specific cell surface receptors, but the signal transduction pathways are largely unresolved as yet. In this study we report that TGF-beta 1 induces a rapid phosphorylation of the cyclic AMP responsive element binding protein (CREB) in mink lung CCl64 cells. Phosphorylation induced by TGF-beta 1 is not mediated by the cAMP-dependent protein kinase. Parallel to the increase in phosphorylation of CREB, an increase in binding to the collagenase TPA responsive element was observed. CREB participates in the binding to this element, probably as a heterodimer with another as yet unknown protein. The modification imposed on CREB and its involvement in an enhanced TRE-binding could be a mechanism by which TGF-beta 1 induces the TRE-mediated transcriptional activation.
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PMID:TGF-beta 1 induces phosphorylation of the cyclic AMP responsive element binding protein in ML-CCl64 cells. 185 Jun 93

Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids encoding PKI(1-31) inhibit the expression that is stimulated by the addition of cAMP analogs in both cell lines; basal expression, however, is inhibited by PKI(1-31) only in the JEG-3 cell line and not in the CV-1 cells. These observations indicate that, in JEG-3 cells, PKI(1-31) is a specific inhibitor of kinase A-mediated gene transcription, but it does not modify kinase C-directed transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells. 247 35

LH has been shown to be the principal hormone regulating ovarian thecal-interstitial cell (TIC) differentiation. It has been well documented that LH stimulates cAMP production and that cAMP analogs mimick the stimulatory actions of LH, but the mechanisms by which LH and cAMP stimulate TIC differentiation are unknown. The purpose of these studies was to characterize LH-stimulated differentiation of isolated TIC in serum-free medium and examine the role of cAMP-dependent protein kinase (PKA) isoenzymes in TIC differentiation. Highly purified (greater than 90%) TIC which were free from granulosa cell contamination were isolated from collagenase-dispersed ovaries of hypophysectomized immature rats by Percoll gradient centrifugation. When the purified TIC (20,000 viable cells/well) were cultured (2 days) in serum-free medium (0.2 ml in 96-well plates), low levels of steroids were produced. LH stimulated a dose-related (ED50 = 2.6 +/- 0.4 ng/ml) increase (50-fold) in androsterone, the principal androgen produced. LH stimulated an immediate dose-related increase in cAMP production, but there was a 20-h lag before LH stimulated an increase in androsterone production, which reached maximum levels at 30 h. LH-stimulated progesterone production increased immediately to a maximum at 10 h, then progesterone levels decreased as androsterone production increased. To determine the role of PKA in stimulating androsterone and progesterone production, TIC were cultured (2 days) with 8-aminohexylamino-cAMP (100 microM) plus N6-benzoyl-cAMP (30 microM) or 8-thiomethyl-cAMP (30 microM) plus N6-benzoyl-cAMP (30 microM) to directly and selectively activate type I or type II PKA, respectively. Selective activation of either isoenzyme increased androsterone and progesterone production by TIC. Immunoblots revealed that either type I or type II PKA increased the contents of P450scc and P45017 alpha in TIC. This is the first demonstration that direct activation of either type I or type II PKA stimulates TIC differentiation. These results indicate that LH stimulates TIC differentiation by a mechanism mediated by activation of one or both PKA isoenzymes.
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PMID:Evidence that luteinizing hormone-stimulated differentiation of purified ovarian thecal-interstitial cells is mediated by both type I and type II adenosine 3',5'-monophosphate-dependent protein kinases. 254 87

The sequence of events within the ovary during the process of ovulation discussed in this review is schematically represented in Fig. 1. It is obvious that LH, perhaps with some contribution from FSH, is the normal physiological trigger for the ovulatory sequence of events, and it appears from the available information that the effects of LH are mainly mediated via adenylate cyclase and increased cAMP levels. The cAMP in turn, via cAMP-dependent protein kinase, influences at least three distinct steps in the ovulatory process which seem to be of crucial importance, namely 1) the stimulation of steroidogenesis; 2) the stimulation of cyclooxygenase/lipooxygenase leading to increased prostaglandin/leukotriene synthesis; and 3) the stimulation of plasminogen activator which catalyzes the conversion of plasminogen to plasmin. A fourth crucial step in the ovulatory mechanism is the LH-induced increase in latent collagenase, but it remains to be determined if this step is mediated via cAMP. Concomitant with the increase in latent collagenase, there also appears to be an LH-dependent increase in collagenase inhibitors. The latent collagenase is then activated, and it appears that leukotrienes and prostaglandins, as well as plasmin, may be involved in this process. The active collagenase causes a digestion of the collagen in the follicle wall, and plasmin, as well as possibly other proteolytic enzymes such as proteoglycanases, may cause a further dissociation of the follicular wall. These processes of digestion of collagen and dissociation of the collagen fibers result in an opening in the follicular wall with the formation of the stigma and rupture. While the weakening of the follicular wall takes place throughout the entire wall, rupture remains for the most part a localized process at the apex of the follicle. This localization of the rupture may be explained on the basis of mechanical factors operating when the follicle wall thins and weakens. While it is clear that prostaglandins and leukotrienes can influence smooth muscle by causing contractions and that these compounds can cause vascular changes such as increased permeability, vasodilation, and vasoconstriction, it is not clear what the exact role of these latter processes are in ovulation. It appears that progesterone and not estrogen play an important role in the mechanism of LH-induced follicular rupture, but the locus of action of progesterone and its mechanism of action remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanism of mammalian ovulation. 255 97

The sequence of ovarian events during the process of ovulation discussed in this review is schematically represented in Figure 1. It is obvious that LH, perhaps with some contribution from FSH, is the normal physiological trigger for the ovulatory sequence of events and it appears from the available information that LH's effects are mainly mediated via adenylate cyclase and increased cAMP. The cAMP in turn, via cAMP-dependent protein kinase, influences at least three distinct steps in the ovulatory process which seem to be of crucial importance, namely 1) the stimulation of steroidogenesis; 2) the stimulation of cyclooxygenase/lipooxygenase leading to increased prostaglandin/leukotriene synthesis; and 3) the stimulation of plasminogen activator which catalyzes the conversion of plasminogen to plasmin. A fourth crucial step in the ovulatory mechanism is the LH-induced increase in latent collagenase, but it remains to be determined if this step is mediated via cAMP. Concomitant with the increase in latent collagenase, there also appears to be an LH-dependent increase in collagenase inhibitors. The latent collagenase is then activated and it appears that leukotrienes and prostaglandins as well as plasmin may be involved in this process. The active collagenase causes a digestion of the collagen in the follicle wall. Plasmin as well as possibly other proteolytic enzymes such as proteoglycanases (Too et al., 1984) may cause a further dissociation of the follicular wall. These processes of digestion of collagen and dissociation of the collagen fibers result in an opening in the follicular wall with the formation of the stigma and rupture. While the weakening of the follicular wall takes place throughout the entire wall, rupture remains for the most part a localized process at the apex of the follicle. This localization of the rupture may be explained on the basis of mechanical factors operating when the follicle wall thins and weakens (Rodbard, 1984). While it is clear that prostaglandins and leukotrienes can influence smooth muscle by causing contractions and that these compounds can cause vascular changes such as increased permeability, vasodilatation and vasoconstriction, it is not clear what the exact role of these latter processes are in ovulation. It appears that progesterone and not estrogen play an important role in the mechanism of LH induced follicular rupture, but the locus of action of progesterone and its mechanism of action remains to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mechanism of mammalian ovulation. 265 83

The amino acid sequences surrounding three major phosphorylation sites in rat and bovine synapsin I have been determined by employing automated gas-phase sequencing and manual Edman degradation of purified phosphopeptide fragments. Site 1 is a serine residue phosphorylated by cAMP-dependent protein kinase and by calcium/calmodulin-dependent protein kinase I. The sequence around site 1 was derived from tryptic/chymotryptic phosphopeptides and overlapping cyanogen bromide cleavage fragments. This sequence, identical in rat and bovine synapsin I, is Asn-Tyr-Leu-Arg-Arg-Arg-Leu-Ser(P)-Asp-Ser-Asn-Phe-Met. Site 1 is located at the NH2 terminus of the protein, within the collagenase-resistant head region. Sites 2 and 3 are serine residues phosphorylated by calcium/calmodulin-dependent protein kinase II. The sequences surrounding bovine site 2 and site 3 were derived from tryptic phosphopeptides and overlapping fragments generated by cleavage with chymotrypsin, collagenase, and endoproteinase Lys-C. The sequence around bovine site 2 is Thr-Arg-Gln-Thr-Ser(P)-Val-Ser-Gly-Gln-Ala-Pro-Pro-Lys, and the sequence around bovine site 3 is Thr-Arg-Gln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Met-Pro-Arg. Sites 2 and 3 are located within the COOH-terminal, collagenase-sensitive tail region of the molecule, separated by 36 amino acids. The sequences surrounding rat site 2 and site 3 were derived from tryptic phosphopeptides. The sequence around rat site 2 is Gln-Ala-Ser(P)-Ile-Ser-Gly-Pro-Ala-Pro-Pro-Lys, and the sequence around rat site 3 is Gln-Ala-Ser(P)-Gln-Ala-Gly-Pro-Gly-Pro-Arg. Thus, the sequences surrounding the four sites that are phosphorylated by calcium/calmodulin-dependent protein kinase II, namely sites 2 and 3 in rat and bovine synapsin I, exhibit a high degree of homology.
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PMID:Amino acid sequences surrounding the cAMP-dependent and calcium/calmodulin-dependent phosphorylation sites in rat and bovine synapsin I. 311 71

Protein I, a specific neuronal phosphoprotein, has previously been shown, using rat brain synaptosome preparations, to contain multiple sites of phosphorylation which were differentially regulated by cAMP and calcium. In the present study, Protein I was purified to homogeneity from rat brain and its phosphorylation was investigated using homogeneous cAMP-dependent protein kinase and a partially purified calcium-calmodulin-dependent protein kinase from rat brain. Employing various peptide mapping techniques, a minimum of three phosphorylation sites could be distinguished in Protein I; the phosphorylated amino acid of each site was serine. One phosphorylation site was located in the collagenase-resistant portion of Protein I and was the principal target for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase. This site was also phosphorylated by calcium-calmodulin-dependent protein kinase. The other two phosphorylation sites were located in the collagenase-sensitive portion of Protein I. These latter sites were markedly phosphorylated by calcium-calmodulin-dependent protein kinase, but not by cAMP-dependent protein kinase in concentrations sufficient to phosphorylate maximally the site in the collagenase-resistant portion. Thus, the phosphorylation of purified Protein I by purified cAMP-dependent and calcium-calmodulin-dependent protein kinases provides an enzymological explanation for the regulation of phosphorylation of endogenous Protein I in synaptosome preparations by cAMP and by calcium observed previously. The studies suggest that certain of the synaptic actions of two distinct second messengers, cAMP and calcium, are expressed through the distinct specificities of cAMP- and calcium-dependent protein kinases for the multiple phosphorylation sites in one neuron-specific protein, Protein I.
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PMID:Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases. 625 98

We have studied the compartmentation of cyclic AMP action in purified ventricular cardiomyocytes prepared by collagenase perfusion of adult rabbit hearts. Incubation of purified adult myocytes with 1 microM isoproterenol causes rapid accumulation of intracellular cyclic AMP in both soluble (2.3 leads to 7.7 pmol/ mg of protein) and particulate (3.0 leads to 9.2) fractions of cell homogenates (3000 X g for 5 min), increases in the total activity and activity ratio of soluble cyclic AMP-dependent protein kinase (0.21 leads to 0.66), a decrease in protein kinase activity remaining in the particulate fraction (47 leads to 30%), and an increase in the activity ratio of glycogen phosphorylase (0.15 leads to 0.47). Incubation of myocytes with 10 microM prostaglandin E1 (PGE1) leads to a comparable increase in soluble cyclic AMP (2.3 leads to 5.8 pmol/mg of protein) and activation of soluble cyclic AMP-dependent protein kinase (0.21 leads to 0.39) but does not result in any change in cAMP or protein kinase in the particulate fraction and fails to cause an activation of glycogen phosphorylase. PGE1 does not inhibit the effects of isoproterenol; when myocytes are incubated with both isoproterenol and PGE1, the accumulation of cyclic AMP, activation of cAMP-dependent protein kinase and phosphorylase b leads to a conversion are equal to that achieved with isoproterenol alone. Perturbation of cellular calcium using the ionophore A23187, verapamil, or high or low extracellular calcium did not alter the ability of isoproterenol to cause activation of particulate cAMP-dependent protein kinase or influence the inability of PGE1 to do so. Activation of adenylate cyclase by forskolin (30 microM) caused immediate activation of both soluble and particulate cAMP-dependent protein kinase leading to rapid activation of phosphorylase. We conclude that the hormonally specific compartmentation of cyclic AMP and cAMP-dependent protein kinase that occurs in intact heart (Hayes, J. S., Brunton, L. L., Brown, J. H., Reese, J. B., and Mayer, S. E. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1570-1574) is not explained on the basis of cellular heterogeneity but has a subcellular basis within the cardiomyocyte.
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PMID:Compartments of cyclic AMP and protein kinase in mammalian cardiomyocytes. 630 96

Certain metabolic properties of hormonally responsive osteogenic sarcoma cells derived from a transplantable rat tumor have been compared with those of related normal rat bone cells. All studies were carried out on cells grown in monolayer culture. Normal rat bone cells derived by repeated collagenase/trypsin digestion of newborn rat calvaria. Bone cells selected for comparison were thought to be osteoblast-like, as judged by enrichment of alkaline phosphatase and adenylate cyclase responsiveness to parathyroid hormone and prostaglandin E2. The adenylate cyclases of the two cell strains were similarly stimulated by a range of prostanoids and their metabolites and analogs. Morphology showed the two cell strains to be similar; the only obvious difference was a multilayering of cells in the sarcoma cultures, while the normal cultures showed abundant extracellular fibril formation which was not seen in the tumor cells. Investigation of the cAMP-dependent protein kinase isoenzymes showed the presence of two forms in both cell types, one eluting at a low salt concentration and the other at a high salt concentration. There was approximately twice the amount of the first isoenzyme compared to the second isoenzyme. The results indicate the usefulness of the two cell strains to elucidate further the molecular mechanisms of action of parathyroid hormone and prostaglandins.
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PMID:Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells. 693 60


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